Abstract: Bacillus siamensis LZ88 is a biocontrol bacterium with strong antagonistic ability against a variety of pathogenic fungi. Previous study indicated that volatile organic compounds (VOCs), 2-methylbutyric acid and 3-methylbutyric acid, produced by B. siamensis LZ88 showed significant inhibitory effects on Alternaria alternata. In the present study, the mechanism of 2-methylbutyric acid and 3-methylbutyric acid on A. alternate were further explored. Additionally, RNA-Seq was used to study the transcriptome of two volatile mixtures on A. alternata, and some significantly differentially expressed genes were verified by qRT-PCR. The results showed that the mixed treatment of the two VOCs could significantly inhibit the growth of A. alternata, and the hyphae were shrinkage and collapse. Through differential expression analysis, a total of 3461 differentially expressed genes (DEGs) were obtained, of which 1714 genes were up-regulated and 1747 genes were down-regulated. Functional annotation and enrichment analysis of the obtained DEGs revealed that various biological functions of A. alternata were affected by VOCs, including glycerophospholipid metabolism, MAPK signaling pathway and the synthesis of polyketides. The results give new insight into the inhibition mechanism of biocontrol bacteria metabolites products to A. alternata.